The present invention relates to the identification of species, and in particular, methods and compositions for distinguishing between bacterial and fungal species and determining the identity of bacterial and fungal pathogens in biological samples.
The detection and identification of microorganisms recovered from clinical specimens or environmental sources is an important aspect of clinical microbiology, as this information is important to physicians in making decisions related to methods of treatment. In order that a particular microorganism is identified correctly and consistently, regardless of the source or the laboratory identifying the organism, reproducible systems for identifying microorganisms are critical. As stated by Finegold, xe2x80x9cThe primary purpose of nomenclature of microorganisms is to permit us to know as exactly as possible what another clinician, microbiologist, epidemiologist, or author is referring to when describing an organism responsible for infection of an individual or outbreakxe2x80x9d (S. Finegold, xe2x80x9cIntroduction to summary of current nomenclature, taxonomy, and classification of various microbial agents,xe2x80x9d Clin. Infect. Dis., 16:597 [1993]).
Classification, nomenclature, and identification are three separate, but interrelated aspects of taxonomy. Classification is the arranging of organisms into taxonomic groups (i.e., taxa) on the basis of similarities or relationships. A multitude of prokaryotic organisms has been identified, with great diversity in their types, and many more organisms being characterized and classified on a regular basis.
Classification has been used to organize the seemingly chaotic array of individual bacteria into an orderly framework. Through use of a classification framework, a new isolate can be more easily be characterized by comparison with known organisms. The choice of criteria for placement into groups is currently somewhat arbitrary, although most classifications are based on phylogenetic relationships. An example of the arbitrariness of bacterial classification is reflected in the genetic definition of a xe2x80x9cspeciesxe2x80x9d as being strains of bacteria that exhibit 70% DNA relatedness, with 5% or less divergence within related sequences (Baron et al., xe2x80x9cClassification and identification of bacteria,xe2x80x9d in Manual of Clinical Microbiology, Murray et al. (eds.), ASM Press, Washington, D.C., pp. 249-264 [1995]).
Generally, identification of a bacterium is based on its overall morphological and biochemical patterns observed in culture. Indeed, this is the primary technique employed today in clinical laboratories. Of course, this approach is flawed by the fact that diverse organisms can have similar morphologies and/or biochemical requirements. Moreover, numerous organisms associated with disease may not be cultured in vitro. Indeed, some do not grow well in traditional in vivo culture systems, such as cell cultures or embryonated eggs, nor in vitro such as various nutrient agars and broths.
What is needed is a more defined system for speciation, and in particular, speciation of bacteria and fungi. Such an approach should be amenable to automation, permitting the approach to be used routinely in a clinical laboratory.
The present invention relates to the identification of microbial species, and in particular, methods and compositions for determining the species for an unknown bacterium (or fungus) in a sample. The methods and compositions of the present invention permit distinguishing between bacterial species (or between fungal species) and determining the identity of bacterial (or fungal) pathogens in biological samples. The present invention contemplates a method of speciation that does not require the sequencing of nucleic acid from biological samples. Instead, the method is based on detection of heretofore unknown uniquely conserved portions of ribosomal nucleic acid, such portions being conveniently revealed by restriction digestion of DNA encoding ribosomal nucleic acid, i.e. rRNA genes.
In one embodiment of the method of the present invention for speciation, the present invention contemplates analysis of one or more so-called Ribosomal operons (xe2x80x9crrnxe2x80x9d) of a clinical isolate, the operon comprising three genes often arranged in the order 16S-23S-5S for prokaryotes (and 18S-5.8S-25S for eukaryotes), with xe2x80x9cspacerxe2x80x9d DNA separating each gene (hereinafter represented by: 5xe2x80x2-16S -spacer-23S-spacer-5S-3xe2x80x2). The present invention contemplates that the analysis of at least one of these operons in an unknown bacterial or fungal species (when evaluated for the xe2x80x9csignature band setsxe2x80x9d of a particular species, the signature bands and methods for determining signature bands herein described) allows for accurate speciation.
It is not intended that the present invention be limited by the technique by which the operons are analyzed. In one embodiment, primers directed to these sequences can be employed in an amplification reaction (such as PCR). On the other hand, these conserved sequences can conveniently be analyzed with restriction enzymes. Specifically, the present invention contemplates digesting bacterial or fungal DNA with one or more restriction enzymes which will produce a piece of nucleic acid which is within (or bounded by) the 5xe2x80x2 and 3xe2x80x2 ends of the operon. The resulting digestion product will be conserved for any given species and can serve as a xe2x80x9csignaturexe2x80x9d for that particular species (other species having one or more signature bands of a different size).
Specific embodiments of such a method include (but are not limited to) digestion with one or more restriction enzymes so as to produce any one of the following digestion products:
5xe2x80x2-16S-spacer-23S-spacer-5S-3xe2x80x2,
5xe2x80x2-16S-spacer-23S-spacer-3xe2x80x2,
5xe2x80x2-16S-spacer-23S-3xe2x80x2,
5xe2x80x2-16S-spacer-3xe2x80x2,
5xe2x80x2-16S-3xe2x80x2,
5xe2x80x2-spacer-23S-spacer-5S-3xe2x80x2,
5xe2x80x2-23S-spacer-5S-3xe2x80x2,
5xe2x80x2-spacer-5S-3xe2x80x2,
5xe2x80x2-5S-3xe2x80x2,
5xe2x80x2-23S-3xe2x80x2
5xe2x80x2-spacer-23S-spacer-3xe2x80x2, or
5xe2x80x2-spacer-23S-3xe2x80x2
The present invention also contemplates a host of variations on the above digestion products by cleaving in the middle of genes and/or in the middle of spacer regions. However, for the convenience of detecting such digestion products by gel electrophoresis, it is preferred that the digestion product (due to the relatively limited resolution level of gel electrophoresis) be at least 200 bp in size (and more preferably between 400 and 3000 bp in size).
In one embodiment, the present invention contemplates digestion of such DNA with restriction enzymes that cut only once in the DNA encoding 16S ribosomal RNA and only once in the DNA encoding 23S ribosomal RNA. In a preferred embodiment, the present invention contemplates digestion of bacterial DNA using a single restriction enzyme which cuts only once in the DNA encoding 16S ribosomal RNA and only once in the DNA encoding 23S ribosomal RNA.
In one embodiment, the present invention contemplates a method for bacterial speciation, comprising: i) isolation of bacterial DNA from a sample, said DNA comprising DNA encoding 16S and 23S rRNA; ii) digestion of said isolated DNA with one or more restriction enzymes under conditions such that restriction fragments are produced, said restriction fragments comprising a first digestion product of said DNA encoding 16S and 23S rRNA, said first digestion product comprising at least a portion of said DNA encoding 16S rRNA and at least a portion of said DNA encoding 23S rRNA; iii) separation of said restriction fragments (e.g. by gel electrophoresis), iv) detection of said first digestion product.
In another embodiment, the present invention contemplates a method for bacterial speciation, comprising: i) isolation of bacterial DNA from a sample; said DNA comprising DNA encoding 16S and 23S rRNA; ii) digestion of said isolated DNA with one or more restriction enzymes under conditions such that restriction fragments are produced, said restriction fragments comprising first and second digestion products (e.g. signature bands) of said DNA encoding 16S and 23S rRNA, said first digestion product being larger than said second digestion product, and comprising at least a portion of said DNA encoding 16S rRNA and at least a portion of said DNA encoding 23S rRNA; iii) separation of said restriction fragments (e.g. by gel electrophoresis), iv) detection of said first and second digestion products.
In yet another embodiment, the present invention contemplates a method for bacterial speciation, comprising: a) providing i) a first biological sample comprising bacterial DNA from a known bacterial species, and ii) a second biological sample comprising bacterial DNA from a bacterium whose species is unknown; b) isolating i) a first preparation of bacterial DNA from said first sample and ii) a second preparation of bacterial DNA from said second sample, said DNA of said first and second preparations comprising DNA encoding 16S and 23S rRNA; c) digesting, in any order, i) said first preparation of isolated DNA with one or more restriction enzymes under conditions such that a first preparation of restriction fragments are produced, said first preparation of restriction fragments comprising a first digestion product, said first digestion product comprising at least a portion of said DNA encoding 16S rRNA and at least a portion of said DNA encoding 23S rRNA, and ii) said second preparation of isolated DNA with one or more restriction enzymes under conditions such that a second preparation of restriction fragments are produced, said second preparation of restriction fragments comprising a second digestion product, said second digestion product comprising at least a portion of said DNA encoding 16S rRNA and at least a portion of said DNA encoding 23S rRNA; d) separating, in any order, i) said restriction fragments (e.g. by gel electrophoresis) from said first preparation, and ii) said restriction fragments (e.g. by gel electrophoresis) from said second preparation; and e) comparing of said first and second digestion products.
It is convenient to isolate bacterial DNA by lysis of bacteria to release DNA. It is also convenient to separate restriction fragments by gel electrophoresis, followed by transfer to a membrane for blotting with an oligonucleotide probe.
It is not intended that the present invention be limited by the nature of the sample. The terms xe2x80x9csamplexe2x80x9d and xe2x80x9cspecimenxe2x80x9d in the present specification and claims are used in their broadest sense. On the one hand they are meant to include a specimen or culture. On the other hand, they are meant to include both biological and environmental samples. These terms encompasses all types of samples obtained from humans and other animals, including but not limited to, body fluids such as urine, blood, fecal matter, cerebrospinal fluid (CSF), semen, and saliva, cells as well as solid tissue (including both normal and diseased tissue). These terms also refers to swabs and other sampling devices which are commonly used to obtain samples for culture of microorganisms. In addition, fluids such as IV fluids, water supplies and the like are contemplates as samples.
It is also not intended that the invention be limited by the particular purpose for carrying out the biological reactions. The present invention is applicable to medical testing, food testing, agricultural testing and environmental testing. In one medical diagnostic application, it may be desirable to simply detect the presence or absence of specific pathogens (or pathogenic variants) in a clinical sample. In yet another application, it may be desirable to distinguish one species or strain from another.
With regard to distinguishing different species, in one embodiment, the present invention contemplates comparing two samples suspected to be different species. In another embodiment, a species that is suspected to have changed or diverged from the parent species is compared with the parent species. For example, a species or strain of bacteria may develop a different susceptibilities to a drug (e.g. antibiotics) as compared to the parent species; rapid identification of the specific species or subspecies aids diagnosis and allows initiation of appropriate treatment.
It is not intended that the present invention be limited by the means of detection or the means of comparing first and second digestion products. In one embodiment, said digestion products that are separated by gel electrophoresis are probed with a labeled oligonucleotide in a hybridization reaction.
The present invention can be used with-particular success when comparing samples. In one embodiment, the present invention contemplates a method of analyzing nucleic acid in biological samples, comprising: a) providing: i) first and second samples comprising bacterial nucleic acid, ii) a restriction enzyme capable of generating a restriction fragment with (or bounded by) the 5xe2x80x2 and 3xe2x80x2 ends of a bacterial Ribosomal operon b) treating said nucleic acid of each of said two samples under conditions so as to produce restriction fragments; c) separating said restriction fragments; and d), comparing said restriction fragments from said first and second samples.
It is not intended that the present invention be limited by the number or nature of samples compared. Clinical, food, agricultural, and environmental samples are specifically contemplated within the scope of the present invention.
The present invention contemplates using restriction enzymes wherein the corresponding restriction enzyme recognition sequence exists only once in the 16s and 23s nucleic acid. Alternatively, restriction enzymes can be selected based on the known nucleic acid sequences (see e.g. FIGS. 4 and 6).
To facilitate understanding of the invention, a number of terms are defined below.
xe2x80x9cNucleic acid sequencexe2x80x9d and xe2x80x9cnucleotide sequencexe2x80x9d as used herein refer to an oligonucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
Prokaryotic ribosomes are constructed from 50S and 30S subunits that join together to form a 70S ribosome. The large subunit comprises a single xe2x80x9c23S rRNAxe2x80x9d molecule and a xe2x80x9c5S rRNAxe2x80x9d molecule, while the small subunit comprises a single xe2x80x9c16S rRNAxe2x80x9d molecule.
As used herein, the terms xe2x80x9ccomplementaryxe2x80x9d or xe2x80x9ccomplementarityxe2x80x9d are used in reference to xe2x80x9cpolynucleotidesxe2x80x9d and xe2x80x9coligonucleotidesxe2x80x9d (which are interchangeable terms that refer to a sequence of nucleotides) related by the base-pairing rules. For example, the sequence xe2x80x9cC-A-G-T,xe2x80x9d is complementary to the sequence xe2x80x9cG-T-C-A.xe2x80x9d
Complementarity can be xe2x80x9cpartialxe2x80x9d or xe2x80x9ctotal.xe2x80x9d xe2x80x9cPartialxe2x80x9d complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules. xe2x80x9cTotalxe2x80x9d or xe2x80x9ccompletexe2x80x9d complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.
Ribosomal RNA molecules are characterized by the presence of numerous sequences that can form complementary base pairs with sequences located else where in the same molecule. Such interactions cause rRNA molecules to fold into three-dimensional configurations that exhibit localized double-stranded regions.
As used herein, the term xe2x80x9cgenexe2x80x9d means the deoxyribonucleotide sequences comprising the coding region and including sequences located adjacent to the coding region on both the 5xe2x80x2 and 3xe2x80x2 ends typically for a distance of about 1-3 kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5xe2x80x2 of the coding region and which are present on the mRNA are referred to as 5xe2x80x2 non-translated sequences. The sequences which are located 3xe2x80x2 or downstream of the coding region and which are present on the mRNA are referred to as 3xe2x80x2 non-translated sequences. The term xe2x80x9cgenexe2x80x9d encompasses both cDNA and genomic forms of a gene.
The chromosomal DNA of prokaryotic cells contains multiple copies of the genes coding for rRNAs. For example, the bacterium E. coli contains seven sets of rRNA genes. In the rRNA transcription unit of E. coli, the three genes are typically arranged in the order 16S-23S-5S, with xe2x80x9cspacerxe2x80x9d DNA separating each gene (the spacer DNA separating 23S from 16S typically comprises one or more tRNA genes in addition to unencoded).
The terms xe2x80x9chomologyxe2x80x9d and xe2x80x9chomologousxe2x80x9d as used herein in reference to nucleotide sequences refer to a degree of complementarity with other nucleotide sequences. There may be partial homology or complete homology (i.e., identity). A nucleotide sequence which is partially complementary, i.e., xe2x80x9csubstantially homologous,xe2x80x9d to a nucleic acid sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence. The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence to a target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.
Low stringency conditions comprise conditions equivalent to binding or hybridization at 42xc2x0 C. in a solution consisting of 5xc3x97SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4.H2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS; 5xc3x97Denhardt""s reagent [50xc3x97Denhardt""s contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 xcexcg/ml denatured salmon sperm DNA followed by washing in a solution comprising 5xc3x97SSPE, 0.1% SDS at 42xc2x0 C. when a probe of about 500nucleotides in length is employed.
Other equivalent conditions may be employed to comprise low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol), as well as components of the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions. In addition, conditions which promote hybridization under conditions of high stringency can be used (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).
When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term xe2x80x9csubstantially homologousxe2x80x9d refers to any probe which can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.
When used in reference to a single-stranded nucleic acid sequence, the term xe2x80x9csubstantially homologousxe2x80x9d refers to any probe which can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.
As used herein, the term xe2x80x9chybridizationxe2x80x9d is used in reference to the pairing of complementary nucleic acids using any process by which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acids.
As used herein the term xe2x80x9chybridization complexxe2x80x9d refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution (e.g., C0t or R0t analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized to a solid support [e.g., a nylon membrane or a nitrocellulose filter as employed in Southern and Northern blotting, dot blotting or a glass slide as employed in in situ hybridization, including FISH (fluorescent in situ hybridization)].
As used herein, the term xe2x80x9cTmxe2x80x9d is used in reference to the xe2x80x9cmelting temperature.xe2x80x9d The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands (the mid-point). The equation for calculating the Tm of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl [see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)]. Other references include more sophisticated computations which take structural as well as sequence characteristics into account for the calculation of Tm.
As used herein the term xe2x80x9cstringencyxe2x80x9d is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. xe2x80x9cStringencyxe2x80x9d typically occurs in a range from about Tmxe2x88x925xc2x0 C. (5xc2x0 C. below the Tm of the probe) to about 20xc2x0 C. to 25xc2x0 C. below Tm. As will be understood by those of skill in the art, a stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences.
As used herein, the term xe2x80x9camplifiable nucleic acidxe2x80x9d is used in reference to nucleic acids which may be amplified by any amplification method. It is contemplated that xe2x80x9camplifiable nucleic acidxe2x80x9d will usually comprise xe2x80x9csample template.xe2x80x9d
As used herein, the term xe2x80x9csample templatexe2x80x9d refers to nucleic acid originating from a sample which is analyzed for the presence of a target sequence of interest. In contrast, xe2x80x9cbackground templatexe2x80x9d is used in reference to nucleic acid other than sample template which may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.
xe2x80x9cAmplificationxe2x80x9d is defined as the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction technologies well known in the art [Dieffenbach C W and G S Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.]. As used herein, the term xe2x80x9cpolymerase chain reactionxe2x80x9d (xe2x80x9cPCRxe2x80x9d) refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195 and 4,683,202, hereby incorporated by reference, which describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. The length of the amplified segment of the desired target sequence is determined by the relative positions of two oligonucleotide primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the xe2x80x9cpolymerase chain reactionxe2x80x9d (hereinafter xe2x80x9cPCRxe2x80x9d). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be xe2x80x9cPCR amplifiedxe2x80x9d.
With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.
Amplification in PCR requires xe2x80x9cPCR reagentsxe2x80x9d or xe2x80x9cPCR materialsxe2x80x9d, which herein are defined as all reagents necessary to carry out amplification except the polymerase, primers and template. PCR reagents nomally include nucleic acid precursors (dCTP, dTTP etc.) and buffer.
As used herein, the term xe2x80x9cprimerxe2x80x9d refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
As used herein, the term xe2x80x9cprobexe2x80x9d refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligpnucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labelled with any xe2x80x9creporter molecule,xe2x80x9d so that it is detectable using any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.
As used herein, the terms xe2x80x9crestriction endonucleasesxe2x80x9d and xe2x80x9crestriction enzymesxe2x80x9d refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence. Such enzymes can be used to create Restriction Fragment Length Polymorphisms (RFLPs). RFLPs are in essence, unique fingerprint snapshots of a piece of DNA, be it a whole chromosome (genome) or some part of this, such as the regions of the genome that specifically flank ribosomal operons. All such RFLP fingerprints are indicative of the random mutations in all DNA molecules that inevitably occur over evolutionary time. Because of this, if properly interpreted, evolutionary relatedness of any two genomes can be compared based on the fundamental assumption that all organisms have had a common ancestor. Thus, the greater the difference in RFLP fingerprint profiles, the greater the degree of evolutionary divergence between them (although there are exceptions). With such an understanding, it then becomes possible, using appropriate algorithms, to covert RFLP profiles of a group of organisms (e.g. bacterial isolates) into a phylogenic (evolutionary) tree.
RFLPs are generated by cutting (xe2x80x9crestrictingxe2x80x9d) a DNA molecule with a restriction endonuclease. Many hundreds of such enzymes have been isolated, as naturally made by bacteria. In essence, bacteria use such enzymes as a defensive system, to recognize and then cleave (restrict) any foreign DNA molecules which might enter the bacterial cell (e.g. a viral infection). Each of the many hundreds of different restriction enzymes has been found to cut (i.e. xe2x80x9ccleavexe2x80x9d or xe2x80x9crestrictxe2x80x9d) DNA at a different sequence of the 4 basic nucleotides (A, T, G, C) that make up all DNA molecules, e.g. one enzymes might specifically and only recognize the sequence A-A-T-G-A-C, while another might specifically and only recognize the sequence G-T-A-C-T-A, etc. etc. Dependent on the unique enzyme involved, such recognition sequences vary in length, from as few as 4 nucleotides (e.g. A-T-C-C) to as many as 21 nucleotides (A-T-C-C-A-G-G-A-T-G-A-C-A-A-A-T-C-A-T-C-G). From here, the simplest way to consider the situation is that the larger the recognition sequence, the fewer restriction fragments will result as the larger the recognition site, the lower the probability is that it will repeatedly be found throughout the genomic DNA.
In one embodiment, the present invention utilizes the restriction enzyme called EcoRI which has a 6 base pair (nucleotide) recognition site. Thus, given that there exist but 4 nucleotides (A,T,G,C), the probability that this unique 6 base recognition site will occur is 46, or once in every 4,096 nucleotides. Given that the H. influenzae (xe2x80x9cHixe2x80x9d) genome (chromosome) is approximately 2xc3x97106 bp (base pairs) in length, digestion of this DNA with EcoRI theoretically should yield 488 fragments. This varies significantly from isolate to isolate of H. influenzae because of xe2x80x9crandom mutationsxe2x80x9d that inevitably occurs over evolutionary time, some of which either destroy an EcoRI sequence cutting site, or create a new one. As such, the overall degree of variation in EcoRI RFLP profiles among a series of isolates within a given species such as H. influenzae, is indicative of the degree of genetic relatedness of these isolates (although there are exception). Using appropriate algorithms, such RFLP profiles are readily converted to xe2x80x9cphylogenetic treesxe2x80x9d (see e.g. FIG. 3) which are simply a diagrammatic figures indicating the evolutionary divergence of isolates from some theoretically common ancestor.
Once the genomic (chromosomal) DNA of a bacterial isolate has been isolated, it is then digested (cut) with an enzyme such as EcoRI. Following the digestion, the resultant individual fragments are separated from one another based on their sizes. This can be done by using agarose gel electrophoresis. In essence, during electrophoresis the smaller molecules (DNA fragments) move faster than larger one and thus the resultant separation is a gradient from the largest to the smallest fragments. These can easily be visualized as bands down the electrophoresis gel, from the top to the bottom with the smallest fragments bottom-most.
Using ribotyping methodology, DNA fragments involving the multiple (e.g. 6 for the case of H. influenzae, 7 for the case of E. coli, etc) ribosomal operons and the immediately flanking DNA sequences (genes) can be distinguished by hybridization of the resultant electrophoresis separated DNA fragments with a radioactively labeled ribosomal operon DNA probe. This then reduces the total number of visualized DNA fragments (predicted above to be approximately 488 restriction fragments) to those only including or immediately flanking the RNA operons, about 14 fragments in toto for H. influenzae. Nonetheless, because of inevitable random background mutation indicative of evolutionary time, with the exception of very recently evolved clones, every independent isolate of H. influenzae will have a variant EcoRI ribotype RFLP profile. And the more variant, the more distantly related will be any two isolates so compared. In contrast, rigorous conservation of 16S and 23S rRNA sequences makes possible the unique species-specific RFLPs produced according to the methods and compositions of the present invention.
DNA molecules are said to have xe2x80x9c5xe2x80x2 endsxe2x80x9d and xe2x80x9c3xe2x80x2 endsxe2x80x9d because mononucleotides are reacted to make oligonucleotides in a manner such that the 5xe2x80x2 phosphate of one mononucleotide pentose ring is attached to the 3xe2x80x2 oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide is referred to as the xe2x80x9c5xe2x80x2 endxe2x80x9d if its 5xe2x80x2 phosphate is not linked to the 3xe2x80x2 oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the xe2x80x9c3xe2x80x2 endxe2x80x9d if its 3xe2x80x2 oxygen is not linked to a 5xe2x80x2 phosphate of another mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5xe2x80x2 and 3xe2x80x2 ends. In either a linear or circular DNA molecule, discrete elements are referred to as being xe2x80x9cupstreamxe2x80x9d or 5xe2x80x2 of the xe2x80x9cdownstreamxe2x80x9d or 3xe2x80x2 elements. This terminology reflects the fact that transcription proceeds in a 5xe2x80x2 to 3xe2x80x2 fashion along the DNA strand.
As used herein, the term xe2x80x9can oligonucleotide having a nucleotide sequence encoding a genexe2x80x9d means a nucleic acid sequence comprising the coding region of a gene, i.e. the nucleic acid sequence which encodes a gene product. The coding region may be present in either a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide may be single-stranded (i.e., the sense strand) or double-stranded.
As used herein, the terms xe2x80x9cnucleic acid molecule encoding,xe2x80x9d xe2x80x9cDNA sequence encoding,xe2x80x9d and xe2x80x9cDNA encodingxe2x80x9d refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.
The term xe2x80x9cSouthern blotxe2x80x9d refers to the analysis of DNA on agarose or acrylamide gels to fractionate the DNA according to size, followed by transfer and immobilization of the DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized DNA is then probed with a labeled oligo-deoxyribonucleotide probe or DNA probe to detect DNA species complementary to the probe used. The DNA may be cleaved with restriction enzymes prior to electrophoresis. Following electrophoresis, the DNA may be partially depurinated and denatured prior to or during transfer to the solid support. Southern blots are a standard tool of molecular biologists [J. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., pp 9.31-9.58].
The term xe2x80x9cNorthern blotxe2x80x9d as used herein refers to the analysis of RNA by electrophoresis of RNA on agarose gels to fractionate the RNA according to size followed by transfer of the RNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized RNA is then probed with a labeled oligo-deoxyribonucleotide probe or DNA probe to detect RNA species complementary to the probe used. Northern blots are a standard tool of molecular biologists [J. Sambrook, J. et al. (1989) supra, pp 7.39-7.52].
The term xe2x80x9creverse Northern blotxe2x80x9d as used herein refers to the analysis of DNA by electrophoresis of DNA on agarose gels to fractionate the DNA on the basis of size followed by transfer of the fractionated DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized DNA is then probed with a labeled oligo-ribonuclotide probe or RNA probe to detect DNA species complementary to the ribo probe used.
The term xe2x80x9cisolatedxe2x80x9d when used in relation to a nucleic acid, as in xe2x80x9can isolated oligonucleotidexe2x80x9d refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is nucleic acid present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA which are found in the state they exist in nature.
As used herein, the term xe2x80x9cpurifiedxe2x80x9d or xe2x80x9cto purifyxe2x80x9d refers to the removal of undesired components from a sample.
As used herein, the term xe2x80x9csubstantially purifiedxe2x80x9d refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated. An xe2x80x9cisolated polynucleotidexe2x80x9d is therefore a substantially purified polynucleotide.
The term xe2x80x9csamplexe2x80x9d as used herein is used in its broadest sense and includes environmental and biological samples. Environmental samples include material from the environment such as soil and water. Biological samples may be animal, including, human, fluid (e.g., blood, plasma and serum), solid (e.g., stool), tissue, liquid foods (e.g., milk), and solid foods (e.g., vegetables).
The term xe2x80x9cbacteriaxe2x80x9d and xe2x80x9cbacteriumxe2x80x9d refer to all prokaryotic organisms, including those within all of the phyla in the Kingdom Procaryotae. It is intended that the term encompass all microorganisms considered to be bacteria including Mycoplasma, Chiamydia, Actinomyces, Streptomyces, and Rickettsia. All forms of bacteria are included within this definition including cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc. Also included within this term are prokaryotic organisms which are gram negative or gram positive. xe2x80x9cGram negativexe2x80x9d and xe2x80x9cgram positivexe2x80x9d refer to staining patterns with the Gram-staining process which is well known in the art [Finegold and Martin, Diagnostic Microbiology, 6th Ed. (1982), C V Mosby St. Louis, pp 13-15]. xe2x80x9cGram positive bacteriaxe2x80x9d are bacteria which retain the primary dye used in the Gram stain, causing the stained cells to appear dark blue to purple under the microscope. xe2x80x9cGram negative bacteriaxe2x80x9d do not retain the primary dye used in the Gram stain, but are stained by the counterstain. Thus, gram negative bacteria appear red.